Abstract 2363 Discovery of a unique Toxoplasma gondii secretory effector protein that relocates ALG-2 and other ESCRT proteins during infection of host cells

The hetero-multimeric protein machinery known as Endosomal Sorting Complex Required for Transport (ESCRT) operates across a spectrum of major cellular pathways including endosomal sorting, intralumenal vesicle biogenesis, and repair of endo-lysosomal and plasma membranes. Because of its pivotal roles in membrane remodeling, the ESCRT machinery is also exploited by many viral and bacterial pathogens for replication and intracellular survival. We recently reported that the protozoan pathogen Toxoplasma gondii (Tg) also subverts the ESCRT machinery, at least in one case for the uptake and degradation of host cytosolic proteins to support of its metabolic needs. Although apoptosis-linked gene 2 (ALG-2) is among the various ESCRT-related proteins that Tg recruits to its intracellular niche, the parasitophorous vacuole (PV), no other pathogens are known to exploit ALG-2; hence, the mechanism behind recruitment of ALG-2 to the PV remained unknown. ALG-2, an EF hand calcium-binding ESCRT accessory protein involved in membrane repair and other functions, binds to other proteins bearing certain short linear amino acid motifs. By searching for Tg secretory proteins containing such motifs, we identified TgGRA8 as a candidate with multiple putative ALG-2 binding elements. Reciprocal immunoprecipitation-mass spectrometry experiments suggested an interaction between TgGRA8 and ALG-2 in infected cells, along with other known ALG-2 binding proteins including ALG-2 interacting protein X (ALIX) and TSG101. Accordingly, by knocking out TgGRA8 we found that it is necessary for ALG-2 and ALIX recruitment to the PV. TgGRA8 also contributes to TSG101 recruitment. Biochemical experiments confirmed a direct and unexpectedly Ca2+-independent interaction between ALG-2 and a peptide from TgGRA8 encompassing one of its three conserved ALG-2 binding elements. Also unexpectedly, binding of GRA8 peptide to ALG-2 generated large oligomers suggestive of the peptide's ability to link ALG-2 molecules together. Collectively, our work identifies TgGRA8 as the first microbial effector targeting host ALG-2. Ongoing studies aim to unravel the biochemical mechanisms of TgGRA8 binding to ALG-2 under native cellular conditions along with defining the specific purpose for which Tg recruits ALG-2 to its PV. We acknowledge the funding support from NIH-NIAID (R01 AI120607) for this research work.